ABSTRACT Improved Detection of Prostate Cancer with New Nanoparticle-based Ultrasound Contrast Agents Targeted to PSMA Prostate cancer (PCa) biopsies are commonly performed using ultrasound (US) guidance, but the delineation of tumors within the prostate with US is not clear. This has led to rising morbidity from current standard of care biopsies that are not aimed at a specific target but rather typical locations where cancer may be found. Imaging of the location of the tumor within the gland and the peri-glandular space would significantly impact the staging of the disease. The development of a new tool to accurately depict cancer within the prostate is thus urgently needed to aid in staging and biopsy guidance. To meet this need, this proposal will develop targeted US contrast agents, which can extravasate to PCa cells and can delineate a target lesion of concern more effectively than current standard of care. Prostate specific membrane antigen (PSMA) is an ideal target to detect PCa due to its abundant expression in most prostate cancers. To provide a better, practical tool for clear identification of PCa for biopsy, we have developed a nano-sized US contrast agent (called a nanobubble ? NB) targeted to PSMA via a new, highly specific small molecule-based ligand. The targeted NBs, are similar in structure to clinically used microbubbles (MB) and are clearly visible on clinical US. In contrast to MB which remain in the vasculature, the <120 nm NB size enables them extravasate and directly bind to cancer cells. This can result in higher accumulation of contrast at the tumor itself leading to better resolution and detection of PCa. The PSMA-targeted agent has the capacity to revolutionize PCa imaging, since US is so broadly available, low cost, and safe. Importantly, US is already frequently utilized in PCa biopsy procedures. The objective of this research is development of a uniquely capable contrast agent for enhanced detection and delineation of PCa with US using two complementary tactics: 1) detection of PSMA on the surface of prostate cancer cells after targeted NB extravasation and 2) monitoring differences in contrast agent dynamics in tumor versus normal prostate tissue. We hypothesize that PSMA-targeted NBs can target PCa cells directly and will thus be more effective at detecting and delineating PCa within and outside of the prostate gland than MBs. We propose four specific aims to support the project objectives: Aim 1 will determine the optimal formulation parameters for the NBs. We seek a stable formulation that will be successfully targeted to PSMA with high yield. The formulation needs to be visible on US and sustain its signal for at least 1 hour to allow for the biopsy procedure to take place. Bubbles will be characterized by dynamic light scattering, confocal microscopy and US. In vitro binding efficacy and cell uptake will be screened in cell culture. Aim 2 will maximize ultrasound NB signal in PSMA positive and negative flank tumors in mice and compare performance to MBs that are clinically available. Aim 3 will examine performance of the NBs in a clinically relevant orthotopic model. We will acquire contrast enhanced images and will then utilize 3D whole mouse cryoimaging to determine the efficacy of segmentation of the tumor and prostate tissue and directly compare histology and location of PCa determined using US imaging. Finally, in Aim 4, we will test the strategy in a dog model of prostate cancer. It is our hope that PSMA-targeted NBs will depict the pattern of primary tumor within or outside the prostate gland and inform planning for biopsy and surgical approaches. Correlation of Pca and US signal will be made in vivo with MRI of the dogs and then confirmed ex vivo using pathology. Improved biopsies and cancer localization will lead to increased detection of high grade tumors and tumor staging, and lower morbidity.